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Article Open Access

circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching

  • Authors:
    • Yujie Wang
    • Xiaoting Li
    • Meirong Wang
    • Nailiang Zhai
    • Mengqi Jiang
    • Bo Liu
    • Changjun Lv
    • Songzi Zhang
    • Xiaodong Song
    • Jinjin Zhang
  • View Affiliations / Copyright

    Affiliations: Department of Cellular and Genetic Medicine, Shandong Key Laboratory of Complex Medical Intelligence and Aging, Shandong Medical and Pharmaceutical University, Yantai, Shandong 264003, P.R. China, Department of Medical Laboratory, Yantai Affiliated Hospital of Shandong Medical and Pharmaceutical University, Yantai, Shandong 264000, P.R. China, Department of Respiratory and Critical Care Medicine, Shandong Medical and Pharmaceutical University Hospital, Shandong Medical and Pharmaceutical University, Binzhou, Shandong 256603, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 260
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    Published online on: July 15, 2026
       https://doi.org/10.3892/ijmm.2026.5931
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Abstract

Pulmonary fibrosis is a progressive and fatal interstitial lung disease characterized by aberrant fibroblast activation and excessive extracellular matrix deposition. Circular RNAs (circRNAs) have emerged as critical regulators of fibrotic pathogenesis. However, their mechanistic roles remain incompletely defined. In the present study, circCACNA1D was identified as a novel driver of pulmonary fibrosis progression through direct interaction with pyruvate kinase M2 (PKM2). Specifically, circCACNA1D binds PKM2 and promotes its nuclear translocation and dimerization. This process is facilitated by desuccinylation at conserved lysine residues (K135, K166 and K270). The conformational shift from the tetrameric to the dimeric state reprograms cellular metabolism toward aerobic glycolysis and activates a pro‑fibrotic transcriptional program, including direct upregulation of KIF4A. In addition, the pharmacological stabilization of PKM2 tetramers with TEPP‑46 attenuated fibrotic phenotypes in vitro and alleviated bleomycin‑induced pulmonary fibrosis in mice. These findings define a previously unrecognized circRNA‑driven axis that coordinates PKM2 conformational switching, metabolic reprogramming and transcriptional activation, and suggest a potential therapeutic strategy for pulmonary fibrosis.
View Figures

Figure 1

Identification of PKM2 as a direct
binding partner of circCACNA1D in pulmonary fibrosis. (A)
Scratch-wound healing assay of MRC-5 fibroblasts treated with
TGF-β1 (5 ng/ml) for 0, 48 and 72 h. (B) Quantitative analysis of
wound closure. (C) Proliferation of MRC-5 cells treated with TGF-β1
for the indicated times was monitored using the IncuCyte S3
live-cell analysis system. (D) Western blot analysis of fibrotic
markers (α-SMA, vimentin and collagen I) in MRC-5 cells stimulated
with TGF-β1 for 48 and 72 h. β-tubulin served as the loading
control. (E) Quantitative analysis of the results of western blot
analysis. (F) Representative immunofluorescence images
demonstrating α-SMA (green) expression in MRC-5 cells following
exposure to TGF-β1 for 0, 48 and 72 h. Nuclei were stained with
DAPI (blue). (G) Silver staining of proteins pulled down by
biotinylated circCACNA1D sense or antisense control probes. (H)
Arrow indicates the specific ~60 kDa band identified by mass
spectrometry. (I) Western blot validation of PKM2 enrichment in the
RNA pull-down assay using a circCACNA1D sense probe. Input lysate
served as the positive control. (J) RIP-qPCR analysis illustrating
the enrichment of circCACNA1D by anti-PKM2 antibody in MRC-5 cells
with or without TGF-β1 (5 ng/ml; 48 h). Normal IgG served as a
negative control. (K) RNA-FISH detection of circCACNA1D (red)
combined with immunofluorescence staining of PKM2 (green) in MRC-5
cells treated with or without TGF-β1. Nuclei were stained with DAPI
(blue; scale bar, 50 μm). *P<0.05,
**P<0.01. PKM2, pyruvate kinase M2; circRNA, circular
RNA; RIP, RNA immunoprecipitation; FISH, fluorescence in
situ hybridization.

Figure 2

circCACNA1D promotes PKM2 nuclear
translocation and dimerization. (A) Nuclear-cytoplasmic
fractionation and western blot analysis of PKM2 in MRC-5 cells
treated with TGF-β1 (5 ng/ml; 48 h), transfected with circCACNA1D
overexpression plasmid (OE circRNA), or transfected with
circCACNA1D knockdown (si-circRNA). Lamin B1 and β-tubulin served
as nuclear and cytoplasmic markers, respectively. (B)
Immunofluorescence staining of PKM2 (red) in MRC-5 cells under the
indicated treatments. Nuclei were stained with DAPI (blue; scale
bar, 50 μm). (C) Chemical cross-linking followed by
non-reducing SDS-PAGE and western blot analysis to assess PKM2
oligomerization in MRC-5 cells treated with or without TGF-β1. (D)
Chemical cross-linking followed by non-reducing SDS-PAGE and
western blot analysis to assess PKM2 oligomerization in MRC-5 cells
transfected with circCACNA1D or an empty vector. Positions of
tetrameric (~240 kDa) and dimeric (~120 kDa) PKM2 are indicated.
PKM2, pyruvate kinase M2; circRNA, circular RNA; NC, negative
control; WT, wild-type.

Figure 3

circCACNA1D promotes PKM2
desuccinylation at specific lysine residues to facilitate dimer
formation. (A) Immunoprecipitation and western blot analysis of
PKM2 succinylation (Ksu, succinyllysine) levels in MRC-5 cells
treated with or without TGF-β1 (5 ng/ml; 48 h). Whole-cell lysate
and IgG controls are shown. (B) Immunoprecipitation and western
blot analysis of PKM2 succinylation (Ksu, succinyllysine) levels in
MRC-5 cells following circCACNA1D overexpression. (C)
Representative MS/MS spectra confirming succinyllysine modification
at K135, K166 and K270 on PKM2 in lung tissues. (D) Sequence
alignment of PKM2 protein sequences from humans and mice,
highlighting conserved lysine residues (K135, K166 and K270)
identified in panel C. (E) Chemical cross-linking followed by
non-reducing SDS-PAGE and western blot analysis to assess the
oligomeric state of WT PKM2 and its succinylation-mimetic mutants
(3KR and 3KE). Positions of tetrameric (~240 kDa) and dimeric (~120
kDa) PKM2 are indicated. (F) Western blot analysis of α-SMA,
vimentin and collagen I in MRC-5 cells transfected with PKM2 WT,
PKM2 (K-R) or PKM2 (K-E). β-tubulin served as a loading control.
WT, wild type; PKM2, pyruvate kinase M2; circRNA, circular RNA.

Figure 4

Pharmacological stabilization of PKM2
tetramers alleviates fibrotic phenotypes in vitro. (A)
Non-reducing SDS-PAGE analysis of PKM2 oligomeric states in
TGF-β1-stimulated MRC-5 cells treated with increasing
concentrations of TEPP-46 (0, 25, 50, 100 and 200 nmol/ml). (B)
Western blot analysis of fibrotic markers (α-SMA, vimentin and
collagen I) in MRC-5 cells treated with TGF-β1 (5 ng/ml) in the
presence or absence of TEPP-46 (50 nmol/ml). (C) Quantitative
analysis of the results of western blot analysis. (D)
Immunofluorescence staining of α-SMA (green) in MRC-5 cells under
the indicated conditions. Nuclei were stained with DAPI (blue;
scale bar, 50 μm). (E) Scratch-wound healing assay of MRC-5
cells treated with TGF-β1 and TEPP-46. (F) Quantitative analysis of
wound closure. (G) Cell proliferation monitored by IncuCyte S3
live-cell analysis under the same conditions.
*P<0.05, **P<0.01. PKM2, pyruvate
kinase.

Figure 5

TEPP-46 attenuates BLM-induced
pulmonary fibrosis in mice. (A) FVC measured on day 28 after BLM
instillation in mice treated with saline (control), BLM alone, BLM
+ vehicle or BLM + TEPP-46. (B) RL assessed under the same
treatment conditions as in (A). (C) Cdyn evaluated under the same
treatment conditions as in (A). (D) Western blot analysis of α-SMA,
vimentin and collagen I protein levels in lung tissue homogenates.
β-tubulin served as a loading control. (E) Immunofluorescence
staining of α-SMA in lung tissue sections (scale bar, 50
μm). (F) Histopathological assessment of lung sections by
H&E staining. (G) Masson's trichrome staining of lung sections
(scale bar, 100 μm). *P<0.05,
**P<0.01. FVC, forced vital capacity; RL, lung
resistance; Cdyn, dynamic compliance; BLM, bleomycin.

Figure 6

Pro-fibrotic function of circCACNA1D
is dependent on PKM2 nuclear dimerization. (A) Scratch-wound
healing assay illustrating the migration of MRC-5 cells
overexpressing circCACNA1D with or without TEPP-46 (50 nmol/ml;
scale bar, 600 μm). (B) Quantitative analysis of wound
closure. (C) Cell proliferation of MRC-5 cells overexpressing
circCACNA1D with or without TEPP-46 (50 nmol/ml), monitored by
IncuCyte S3. (D) Western blot analysis of fibrotic markers (α-SMA,
vimentin and collagen I) under the same conditions as in panel A.
(E) Scratch-wound healing assay under TGF-β1 stimulation following
circCACNA1D knockdown and rescue with PKM2-NLS. (F) Quantitative
analysis of wound closure. (G) Cell proliferation under TGF-β1
stimulation following circCACNA1D knockdown and PKM2-NLS rescue.
(H) Western blot analysis of fibrotic markers under the same
experimental groups as those in panel E. (I) PK activity measured
in MRC-5 cells overexpressing circCACNA1D or an empty vector
control, with or without TEPP-46 (50 nmol/ml) for 48 h. (J) Lactate
secretion in the cell culture supernatant under the same conditions
as those in panel I. (K) PK activity assessed in MRC-5 cells
treated with TGF-β1 (5 ng/ml; 48 h) with or without circCACNA1D
knockdown,and rescued by PKM2-NLS overexpression. (L) Lactate
secretion measured under the same treatment groups as in those in
panel K. *P<0.05, **P<0.01. circRNA,
circular RNA; PK, pyruvate kinase; PKM2-NLS, nuclear-localized
PKM2.

Figure 7

Nuclear PKM2 dimer drives a
pro-fibrotic transcriptional program via direct activation of
KIF4A. (A) Volcano plot of differentially expressed genes between
control vs. TGF-β1 (left) and TGF-β1 + vehicle vs. TGF-β1 + TEPP-46
(right). (B) Venn diagram demonstrating overlap between
TGF-β1-upregulated (529) and TEPP-46-downregulated (389) genes,
yielding 42 candidates. (C) GO enrichment analysis of the 42
overlapping genes. (D) KEGG pathway enrichment analysis of the same
gene set. (E) qPCR validation of candidate gene expression in cells
overexpressing PKM2-NLS. (F) qPCR analysis of KIF4A mRNA levels
under the indicated treatments. (G) Western blot analysis of KIF4A
protein levels under the same conditions as those in panel F. (H)
Western blot analysis of KIF4A protein levels under PKM2-NLS
overexpression. (I) qPCR analysis of KIF4A mRNA levels with TEPP-46
treatment. (J) Western blot analysis of KIF4A protein levels under
the same conditions as those in panel I. (K) Western blot analysis
of fibrotic markers (α-SMA, vimentin and collagen I) in MRC-5 cells
overexpressing PKM2-NLS with or without concurrent KIF4A knockdown
(siKIF4A). (L) Western blot analysis of fibrotic markers (α-SMA,
vimentin and collagen I) in MRC-5 cells overexpressing KIF4A or an
empty vector control. β-tubulin served as a loading control. (M)
ChIP-qPCR showing PKM2 enrichment at the KIF4A promoter region in
TGF-β1-stimulated MRC-5 cells. *P<0.05,
**P<0.01. PKM2, pyruvate kinase; KIF4A, kinesin
family member 4A; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of
Genes and Genomes; PKM2-NLS, nuclear-localized PKM2; ChIP-qPCR,
chromatin immunoprecipitation quantitative PCR.
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Copy and paste a formatted citation
Spandidos Publications style
Wang Y, Li X, Wang M, Zhai N, Jiang M, Liu B, Lv C, Zhang S, Song X, Zhang J, Zhang J, et al: circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching. Int J Mol Med 58: 260, 2026.
APA
Wang, Y., Li, X., Wang, M., Zhai, N., Jiang, M., Liu, B. ... Zhang, J. (2026). circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching. International Journal of Molecular Medicine, 58, 260. https://doi.org/10.3892/ijmm.2026.5931
MLA
Wang, Y., Li, X., Wang, M., Zhai, N., Jiang, M., Liu, B., Lv, C., Zhang, S., Song, X., Zhang, J."circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching". International Journal of Molecular Medicine 58.3 (2026): 260.
Chicago
Wang, Y., Li, X., Wang, M., Zhai, N., Jiang, M., Liu, B., Lv, C., Zhang, S., Song, X., Zhang, J."circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching". International Journal of Molecular Medicine 58, no. 3 (2026): 260. https://doi.org/10.3892/ijmm.2026.5931
Copy and paste a formatted citation
x
Spandidos Publications style
Wang Y, Li X, Wang M, Zhai N, Jiang M, Liu B, Lv C, Zhang S, Song X, Zhang J, Zhang J, et al: circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching. Int J Mol Med 58: 260, 2026.
APA
Wang, Y., Li, X., Wang, M., Zhai, N., Jiang, M., Liu, B. ... Zhang, J. (2026). circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching. International Journal of Molecular Medicine, 58, 260. https://doi.org/10.3892/ijmm.2026.5931
MLA
Wang, Y., Li, X., Wang, M., Zhai, N., Jiang, M., Liu, B., Lv, C., Zhang, S., Song, X., Zhang, J."circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching". International Journal of Molecular Medicine 58.3 (2026): 260.
Chicago
Wang, Y., Li, X., Wang, M., Zhai, N., Jiang, M., Liu, B., Lv, C., Zhang, S., Song, X., Zhang, J."circCACNA1D drives pulmonary fibrosis by regulating pyruvate kinase M2 dimer‑tetramer switching". International Journal of Molecular Medicine 58, no. 3 (2026): 260. https://doi.org/10.3892/ijmm.2026.5931
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